Degradation of eukaryotic mRNAs plays an essential role in modulation of gene expression and in quality control of mRNA biogenesis. Deadenylation is the first major step in mammalian mRNA degradation and is often a rate-limiting step for mRNA decay and translational silencing, making it an important control point for both processes. Yet, the mechanisms by which deadenylation is regulated remain unknown. Mammalian deadenylation proceeds in two phases mediated consecutively by the Pan2-Pan3 and the Ccr4-Caf1 deadenylase complexes. Recent findings that Pan3 and Tob, two poly(A)-binding protein (PABP)-interacting proteins, modulate deadenylation suggest a new mode of gene regulation via changes in deadenylation, which helps cells reset their protein production profile. The importance of deadenylation in regulation of gene expression is further emphasized by recent observations. First, microRNAs (miRNAs) mediate rapid decay of their mRNA targets by accelerating deadenylation as a major route to gene silencing, though the underlying mechanisms and regulation remain largely unclear. Second, non-translatable mRNA-protein complexes (mRNPs) are found in RNA processing bodies (P-bodies), newly discovered cytoplasmic domains implicated in mRNA turnover, storage of non-translatable mRNPs, and translation repression. One major consequence of deadeny- lation is formation of non-translatable mRNPs, and we have shown that deadenylation is required for P- body formation in mammalian cells. It is plausible that deadenylation induces major mRNP remodeling that determines how mRNPs interact with the machinery mediating their subcellular localization, translation, or decay. The specific aims of this proposal focus on addressing the following key questions: 1) What are the mechanisms by which deadenylation is regulated and how does the regulation of deadenylation affect the fate of mRNA?;2) What trans-acting factors are involved in miRNA-mediated deadenylation and decay and how is miRNA-mediated mRNA decay regulated?;and 3) What changes does deadenylation trigger in mRNA-protein or mRNP complexes and how do these changes influence an mRNP's fate? The proposed studies will reveal fundamental principles that govern mammalian mRNA turnover and provide crucial new mechanistic insights into several key issues related to the dynamic relationship between deadenylation, mRNA decay, translation and mRNP remodeling. PUBLIC HEALTH RELEVANCE: The proposed studies will reveal fundamental principles that govern mammalian mRNA turnover and translation. Since many human diseases (e.g., cancers, autoimmune diseases, allergic inflammation, etc.) are associated with an alteration in the level of gene expression controlled by mRNA stability or translation, the proposed study has the potential to unravel new mechanisms underlying these conditions and thus facilitate the development of novel therapeutic agents.